大腸直腸癌之PCDH10基因表現之研究

Abstract

PCDH10全名為Protocadherin 10，屬於Cadherin superfamily中的Protocadherin群。Protocadherin群在大腦神經系統表現顯著，可能參與神經發育與突觸連結。PCDH10有兩種RNA產物，分別為variant 1及variant 2，目前已經有研究於鼻咽癌以及血癌的證據支持此基因之variant 1產物可能與癌症相關。 本論文之目的為研究PCDH10之基因表現，包含PCDH10 variant 1及PCDH10 variant 2兩種mRNA於大腸直腸細胞株、大腸息肉、正常黏膜組織與腫瘤組織，觀察其是否有可能與大腸直腸癌相關，並進行PCDH10 variant 2 cloning。將剔除終止密碼子之conserved coding sequence接入真核表現載體pcDNA3.1/V5-His-TOPO vector。以期在未來能夠進行variant 2之蛋白功能研究。 本論文以反轉錄聚合酶連鎖反應（RT-PCR）檢測PCDH10 variant 1基因表現。8例周邊血液單核細胞檢體僅3例有PCDH10 variant 1 mRNA微弱表現。在12株大腸直腸癌細胞株中僅HCT 116、HT 29測得微弱表現，另外8種癌症細胞株中於MCF7、Hep3B、SK-Hep-1、PC-3、U-937測得表現，除了MCF7、HEK 293測得顯著的表現，其他的細胞表現都很微弱，此外人類胚胎腎臟細胞HEK 293和胎盤組織都有顯著PCDH10 variant 1表現。23對大腸直腸癌之正常黏膜組織和腫瘤組織當中，半定量分析RT-PCR結果，11對檢體（47.83%）之PCDH10 variant 1 mRNA表現於腫瘤組織較正常黏膜組織降低50%以上。 PCDH10 variant 2可測得基因表現於MCF7、HEK 293，而不表現於PBMC及其他細胞株。6對大腸直腸癌檢體中，有4對測得表現於正常組織，1對表現於腫瘤組織，和1對不表現於任一組織，而在轉移至大腸之胰臟癌檢體對中表現於腫瘤組織。息肉組織皆未測得PCDH10 variant 2。 PCDH10 variant 1之即時反轉錄聚合酶連鎖反應（real-time RT-PCR），使用TATA box binding protein（TBP）作為internal control進行相對定量，19對RNA品質良好的檢體中有15對（78.95%）腫瘤組織較正常黏膜組織下降50%以上，和1對原位癌有顯著的降低。19對共有17例息肉組織相較正常黏膜組織呈現表現降低。 PCDH10基因有variant 1、variant 2兩種mRNA，而PCDH10 variant 2僅在美國國家衛生研究院生物科技資訊中心資料庫中有序列資料而沒有研究文獻，所以為了確認RT-PCR測得之PCDH10 variant 2表現並非來自pre-mRNA，利用北方點墨法分析同時表現兩種mRNA之MCF7細胞株，證實PCDH10 variant 2存在，並利用西方點墨法分析MCF7、HEK293、胎盤組織及大腸正常黏膜組織是否表現蛋白質，並且使用轉殖PCDH10 variant 1之HEK 293作為陽性對照細胞，僅轉殖之HEK 293測得顯著蛋白表現，而其他檢體皆未測得蛋白表現。 本論文顯示PCDH10 variant 1在息肉組織和大腸直腸腫瘤檢體較正常黏膜組織顯著降低表現，可能為大腸直腸癌之相關抑癌基因。PCDH10 variant 2則傾向表現在PCDH10 variant 1大量表現之檢體，可能是轉錄反應之副產物，但於1對大腸直腸檢體和1對轉移至大腸之胰臟癌當中，PCDH10 variant 2也能測得於PCDH10 variant 1表現較低之腫瘤檢體而非正常黏膜組織，因此可能還有其他機制影響PCDH10 variant 2表現。

PCDH10 means protocadherin 10, a member of protocadherin family, which was a subgruop of cadherin superfamily. Protocadherin family members were predominantly expressed in the brain, and might play a role in neuron development or synapses connection. PCDH10 had two kinds of mRNA products, namely variant 1 and variant 2 respectively. PCDH10 variant 1 had been implied to be a tumor suppressor gene in multiple haematologic malignancies and nasopharyngeal carcinoma. However, there was no research about variant 2. To study if PCDH10 could be a tumor suppressor gene in colorectal carcinoma, the gene expression in peripheral blood mononuclear cells (PBMC), cell lines and colorectal cancer patients’ paired tissues were analyzed by RT-PCR. PCDH10 variant 1 mRNA was expressed in three of eight PBMC samples, and in HCT 116, HT 29 of the 12 colorectal cancer cell lines, and in MCF7, Hep3B, SK-Hep-1, PC-3, U-937 of the 8 other cancer cell lines. Human kidney embryonic cell HEK 293 and placenta tissues had expression, too. Except for high expression in MCF7 and HEK 293, the other cells had low gene expression level. In 23 paired colorectal patients’ tissues, PCDH10 variant 1 was more than 50% down regulated in 11 tumor tissues (47.83%) than the paired normal mucosa by semi-quantitative analysis. PCDH10 variant 2 was expressed in MCF7, HEK 293 and placenta tissues but not PBMC or the other cell lines. PCDH10 variant 2 was only analyzed in six pairs of colorectal tissues and 1 paired pancreas cancer tissues. In the paired colorectal tissues, except for no expression in one paired tissue, PCDH10 variant 2 was expressed in four normal parts, and one tumor part. And in the paired pancreas cancer tissues, PCDH10 varaint 2 was expressed in tumor part. In addition, PCDH10 variant 2 was not expressed in 12 polyp tissues. PCDH10 variant 1 was subsequent analyzed by real-time RT-PCR, and TATA box binding protein (TBP) as a internal control. PCDH10 variant 1 was down regulated in 15 of the 19 paired tissues (78.95%), and the tumor parts were more than 50% down regulated than the normal parts. In addition, in 1 pairs of benign tumors was significant down regulated, too. And 17 polyps were significant down regulated compared to normal mucosa tissues. Northern blot was performed and showed the existence of mRNA variants in MCF7 cell line. And then western blot was done to check protein expression in MCF7, HEK 293, placenta tissues and normal mucosa samples. HEK 293 transfected with PCDH10 variant 1 was a positive control. In northern blot, PCDH10 variant 2 from MCF7 had been proven existence. However, in western blot, except for the positive control, the protein existence had not been detected in other samples. The significant down regulation in gene expression assays of PCDH10 variant 1 suggests that PCDH10 variant 1 is a tumor suppressor gene of colorectal cancer. PCDH10 variant 2 trends to be detected in samples that have high expression of PCDH10 variant 1, so PCDH10 variant 2 may be a byproduct which results from transcription error. However, PCDH10 variant 2 also can be detected in the tumor part of one pair of colorectal tissues and the paired pancreas cancer tissues that have low PCDH10 variant 1 expression, so the regulation of the expression of PCDH10 variant 2 may involve other mechanisms.